Fluidic cartridge for cytometry and additional analysis

ABSTRACT

The disclosure relates to devices and methods for analyzing particles in a sample. In various embodiments, the present disclosure provides devices and methods for cytometry and additional analysis. In various embodiments, the present disclosure provides a cartridge device and a reader instrument device, wherein the reader instrument device receives, operates, and/or actuates the cartridge device. In various embodiments, the present disclosure provides a method of using a device as disclosed herein for analyzing particles in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/211,175, filed on Dec. 5, 2018, which is a continuation of U.S.patent application Ser. No. 15/803,133, filed on Nov. 3, 2017, thedisclosure of which are incorporated by reference herein in theirentireties.

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/497,075, filed on Nov. 7, 2016, entitled “Fluidic Cartridge forCytometry and Additional Analysis”, the entire contents of which areincorporated herein by reference and relied upon.

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/176,729, filed on Jun. 8, 2016, entitled “Fluidic Units andCartridges for Multi-Analyte Analysis”, which claims priority to U.S.Provisional Patent Application No. 62/174,776, filed on Jun. 12, 2015,the entire contents of each of which are incorporated herein byreference and relied upon.

This application is related to International ApplicationPCT/US2016/036426, filed on Jun. 8, 2016, entitled “Fluidic Units andCartridges for Multi-Analyte Analysis”, which claims priority to U.S.Provisional Patent Application No. 62/174,776, filed on Jun. 12, 2015,the entire contents of each of which are incorporated herein byreference and relied upon.

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/209,226, filed on Jul. 13, 2016, entitled “Volume Sensing inFluidic Cartridge”, which claims priority to U.S. Provisional PatentApplication No. 62/192,488, filed on Jul. 14, 2015, the entire contentsof each of which are incorporated herein by reference and relied upon.

This application is related to International ApplicationPCT/US2016/042089, filed on Jun. 13, 2016, entitled “Volume Sensing inFluidic Cartridge”, which claims priority to U.S. Provisional PatentApplication No. 62/192,488, filed on Jul. 14, 2015, the entire contentsof each of which are incorporated herein by reference and relied upon.

FIELD OF THE DISCLOSURE

The disclosure relates to medicine and cytometry.

BACKGROUND

All publications cited herein are incorporated by reference in theirentirety to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The following description includesinformation that may be useful in understanding the present disclosure.It is not an admission that any of the information provided herein isprior art or relevant to the presently disclosure, or that anypublication specifically or implicitly referenced is prior art.

Flow cytometry is a popular tool for cellular analysis of biologicalsamples. Typical cytometry analyses involve two parts. The first part issample preparation. For example, some cytometry analyses label targetcells with a specific fluorophore, so that these cells can be detectedby an optical measurement of fluorescence signals. In another example,some cytometry analyses require selectively lysing cells in samples,leaving only target cells intact for cytometry measurement. The secondpart is sample analysis. Usually, the sample stream is focused into anarrow stream when flowing through a flow cell, where the target cellsare measured one by one for optical or other signals. This narrow samplestream is usually obtained by hydrodynamic focusing of sheath flow.

The signal measured in flow cytometry can be used to evaluate individualtarget cells' characteristics, such as cell size and cell surfaceroughness. With the help of fluorescent labeling, additional cellularcharacteristics can also be evaluated such as the existence of acellular nucleus, the amount of DNA inside the cell, antigens on acellular membrane, and many other characteristics. As the cells aremeasured one by one, the total number of target cells detected can alsobe determined by counting the number of measured signal peaks.Additionally, some cytometry analyses also require measuring particledensity in the sample, meaning the number of target particles per samplevolume, which is also known as the absolute count in cytometry analyses.For this measurement, not only the total number of detected particlesneeds to be determined, but also the corresponding volume of the sampleneeds to be determined. These two pieces of information can be usedtogether to calculate the number of particles per sample volume, e.g.,the absolute count.

In conventional flow cytometry analyses, the sample preparation stepsare usually carried out by manual operation. For example, thepreparation steps are often performed in different containers, such ascentrifugal tubes or vials, and only the final prepared sample is thenloaded into a commercial cytometer for optical or other measurement.These manual steps of sample preparation require precise fluid handlingby trained technicians and are thus not suitable for applications whereusers are minimally trained.

Furthermore, for applications such as the point-of-care testing inmedical diagnostics, the cytometry analyses are performed in anon-laboratory environment, such as in emergency rooms or physicianoffices. Therefore, it is important that the biological sample isself-contained and not exposed to environment causing biologicalcontaminations. For this purpose, it is advantageous that both thesample preparation step and the measurement step are carried out in aself-contained manner such as inside a non-exposed container.

Additionally, the absolute count measurement requires that the totalnumber of detected target cells and corresponding sample volume beknown. In conventional cytometry analyses, a fixed amount of sample witha known volume is injected into the system to determine the absolutecount. However, the fluidic system often introduces dead volumes,meaning that some portion of the sample does not go through thecytometer measurement. These dead volumes cause the real sample volumebeing measured to be different from the known volume being injected intothe system, and therefore introduce inaccuracy to the absolute count.

With above considerations, there is a need to develop a fluidiccartridge that can perform the cytometry analysis in a self-contained,automated manner, including both the sample preparation and sampleanalysis steps. There is also a need that such a fluidic cartridge canperform not only a cytometry analysis and cell count, but alsoaccurately measure the absolute count.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with devices, systems and methods which aremeant to be exemplary and illustrative, not limiting in scope.

The present disclosure provides various fluidic cartridges and methodsof using and making such fluidic cartridges. These fluidic cartridgescan perform both the sample preparation and the cytometer analysis.These fluidic cartridges can be used for various types of cytometryanalyses. In various embodiments, the fluidic cartridges as disclosedherein can be used to determine the absolute count. In variousembodiments, the fluidic cartridges as disclosed herein can be used forDNA analysis of cell populations in tumor diagnosis. In variousembodiments, the fluidic cartridges as disclosed herein can be used forCD4+/CD8+ lymphocyte subtype analyses in AIDS diagnosis. In variousembodiments, the fluidic cartridges as disclosed herein can be used forcell analysis in complete blood count (CBC). These fluidic cartridgescan also be used for other types of analyses including, but not limitedto, analyzing analytes, proteins, enzymes, nucleic acids, and otherbiological markers in samples.

Various embodiments of the present disclosure provide a device foranalyzing target particles in a sample. In various embodiments, thedevice comprises a cartridge device. In various embodiments, thecartridge device comprises: an inlet configured for receiving the sampleinto the cartridge device; a fluidic structure fluidly connected to theinlet and configured for mixing at least a portion of the sample with atleast a portion of a reagent to form one or more sample mixtures; a flowcell fluidly connected to the fluidic structure and configured forforming one or more sample streams from the one or more sample mixtures,wherein the sample streams are formed in the flow cell without a sheathflow, and wherein the flow cell comprises an optically transparent areaconfigured for measuring an optical signal from the sample streams todetect the target particles in the sample; and a flow sensor fluidlyconnected to the flow cell and configured for measuring a sensing signalfrom the sample streams that enter the flow sensor. In variousembodiments, a cartridge device as disclosed herein further comprises areagent.

In various embodiments, a device as disclosed herein further comprises areader instrument device, wherein the reader instrument device isconfigured for receiving, operating, and/or actuating the cartridgedevice. In various embodiments, the reader instrument device neitherreceives any liquid from the cartridge device nor transfers any liquidinto the cartridge device.

Various embodiments of the present disclosure provide a method foranalyzing target particles in a sample. The method comprises: applyingthe sample to a cartridge device as disclosed herein, which isconfigured for collecting a predetermined sample volume into thecartridge device; transferring the cartridge device into a readerinstrument device as disclosed herein; mixing at least a portion of thecollected sample and at least a portion of a reagent to form one or moresample mixtures inside the cartridge device; forming one or more samplestreams from the one or more sample mixtures in a flow cell inside thecartridge device, wherein the sample streams are formed in the flow cellwithout a sheath flow; measuring an optical signal from the samplestreams at the flow cell to detect the target particles in the samplestreams; and using the reader instrument device to analyze the measuredoptical signal to quantify the target particles in the sample.

Various embodiments of the present disclosure provide a method foranalyzing particles in a sample. The method comprises: applying thesample to a cartridge device as disclosed herein, which is configuredfor collecting a predetermined sample volume into the cartridge device;transferring the cartridge device into a reader instrument device asdisclosed herein; mixing at least a portion of the collected sample andat least a portion of a reagent to form one or more sample mixturesinside the cartridge device; forming one or more sample streams from theone or more sample mixtures in a flow cell inside the cartridge device,wherein at least two separate sample mixtures are transferred into thesame flow cell to form at least two separate sample streams without asheath flow; measuring an optical signal from the sample streams at theflow cell to detect the target particles in the sample streams; andusing the reader instrument device to analyze the measured opticalsignal to quantify the target particles in the sample.

In various embodiments, a method as disclosed herein further comprises:flowing the sample streams through a flow sensor that is fluidlyconnected to the flow cell; measuring a sensing signal from the samplestreams at the flow sensor to detect the entrance of the sample streamsinto the flow sensor and/or the exit of the sample streams out of theflow sensor; and using the reader instrument device to analyze themeasured optical signal and sensing signal to determine theconcentration of the target particles in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of the basic fluidic unit used in acartridge device as disclosed herein.

FIGS. 2A-2D illustrate, in accordance with various embodiments of thedisclosure, a few non-limiting examples of passive valves.

FIGS. 3A-3C illustrate, in accordance with various embodiments of thedisclosure, a few non-limiting examples of active valves.

FIGS. 4A-4C illustrate, in accordance with various embodiments of thedisclosure, one non-limiting example of implementing a passive valve ina basic fluidic unit.

FIG. 5 illustrates, in accordance with various embodiments of thedisclosure, a symbol drawing that represents a basic fluidic unit asdescribed herein.

FIGS. 6A-6B illustrate, in accordance with various embodiments of thedisclosure, one non-limiting example of a sheathless flow cell asdescribed herein and its symbolic drawing.

FIGS. 7A-7B illustrate, in accordance with various embodiments of thedisclosure, one non-limiting example of a flow sensor as describedherein, which has two sensing zones along the length of a fluidicchannel, and its symbolic drawing.

FIGS. 8A-8B illustrate, in accordance with various embodiments of thedisclosure, another non-limiting example of a flow sensor as describedherein, which has only one sensing zone along the length of a fluidicchannel, and its symbolic drawing.

FIGS. 9A-9C illustrate, in accordance with various embodiments of thedisclosure, one exemplary configuration of a cartridge device asdisclosed herein, where a basic fluidic unit 9001, a sheathless flowcell 9007 and a flow sensor 9009 with two sensing zones 9011 and 9012are connected in serial by fluidic conduits 9006 and 9008.

FIGS. 10A-10B illustrate, in accordance with various embodiments of thedisclosure, another exemplary configuration of a cartridge device asdisclosed herein, where a flow sensor 10007 is connected to themicrofluidic channel 10004 of a basic fluidic unit 10001 with a fluidicconduit 10006.

FIGS. 11A-11B illustrate, in accordance with various embodiments of thedisclosure, another exemplary configuration of a cartridge device asdisclosed herein, where a basic fluidic unit 11001, a sheathless flowcell 11007 and a flow sensor 11009 with one sensing zone 11012 areconnected in serial by fluidic conduits 11006 and 11008.

FIGS. 12A-12G illustrate, in accordance with various embodiments of thedisclosure, another exemplary configuration of a cartridge device asdisclosed herein, where two basic fluidic units 12101 and 12201 are usedin serial with a sheathless flow cell 12301 and a flow sensor 12401.

FIGS. 13A-13C illustrate, in accordance with various embodiments of thedisclosure, another exemplary configuration of a cartridge device asdisclosed herein, where three basic fluidic units 13101, 13201 and 13301are used in serial with a sheathless flow cell 13401 and a flow sensor13501.

FIGS. 14A-14B illustrate, in accordance with various embodiments of thedisclosure, another exemplary configuration of a cartridge device asdisclosed herein, where four basic fluidic units 14101, 14201, 14301 and14401 are used in serial with a sheathless flow cell 14501 and a flowsensor 14601.

FIGS. 15A-15D illustrate, in accordance with various embodiments of thedisclosure, the top view (in x-y plane) of a few examples of a flow cellas described herein.

FIGS. 16A-16B illustrate, in accordance with various embodiments of thedisclosure, an example where a plurality of particles flow through aflow cell for detection.

FIGS. 17A-17D illustrate, in accordance with various embodiments of thedisclosure, exemplary designs for determining the absolute count ofparticles, where the outlet 17103 of the flow cell 17101 is coupled tothe inlet 17202 of the flow sensor 17201 by a fluidic conduit 17001.

FIGS. 18A-18B illustrate, in accordance with various embodiments of thedisclosure, another exemplary design for determining the absolute countof particles, where the inlet 18102 of the flow cell 18101 is coupled tothe outlet 18203 of the flow sensor 18201 by a fluidic conduit 18001.

FIG. 19 illustrates, in accordance with various embodiments of thedisclosure, one non-limiting example of an analyzer having a cartridgedevice and a reader instrument device. The cartridge 19101 having thefluidic structure 19102 can be inserted into a docking slot 19202 on thereader instrument 19201.

FIGS. 20A-20B illustrate, in accordance with various embodiments of thedisclosure, exemplar processes for building a sheathless flow cell asdescribed herein.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technical,and scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. Tabelling, Introduction to Microfluidics reprint edition,Oxford University Press (2010); Hguyen et al., Fundamentals andApplications of Microfluidics 2nd ed., Artech House Incorporated (2006);Berg et al., Microfluidics for Medical Applications, Royal Society ofChemistry (2014); Gomez et al., Biological Applications of Microfluidics1st ed., Wiley-Interscience (2008); and Colin et al., Microfluidics 1sted., Wiley-ISTE (2010), provide one skilled in the art with a generalguide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present disclosure. Other features and advantages of thedisclosure will become apparent from the following detailed description,taken in conjunction with the accompanying drawings, which illustrate,by way of example, various features of embodiments of the disclosure.Indeed, the present disclosure is in no way limited to the methods andmaterials described. For convenience, certain terms employed herein, inthe specification, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. It should be understood that this disclosure is not limited tothe particular methodology, protocols, and reagents, etc., describedherein and as such can vary. The definitions and terminology used hereinare provided to aid in describing particular embodiments and are notintended to limit the claims.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areuseful to an embodiment, yet open to the inclusion of unspecifiedelements, whether useful or not. It will be understood by those withinthe art that, in general, terms used herein are generally intended as“open” terms (e.g., the term “including” should be interpreted as“including but not limited to,” the term “having” should be interpretedas “having at least,” the term “includes” should be interpreted as“includes but is not limited to,” etc.).

Unless stated otherwise, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of claims) can be construedto cover both the singular and the plural. The recitation of ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range.Unless otherwise indicate herein, each individual value is incorporatedinto the specification as if it were individually recited herein. Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (for example,“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the application and does not pose alimitation on the scope of the application otherwise claimed. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.” No language in thespecification should be construed as indicating any non-claimed elementessential to the practice of the application.

Various embodiments of the present disclosure provide a device foranalyzing target particles in a sample. In various embodiments, thedevice comprises a cartridge device. In various embodiments, thecartridge device comprises: an inlet configured for receiving the sampleinto the cartridge device; a fluidic structure fluidly connected to theinlet and configured for mixing at least a portion of the sample with atleast a portion of a reagent to form one or more sample mixtures; a flowcell fluidly connected to the fluidic structure and configured forforming one or more sample streams from the one or more sample mixtures,wherein the sample streams are formed in the flow cell without a sheathflow, and wherein the flow cell comprises an optically transparent areaconfigured for measuring an optical signal from the sample streams todetect the target particles in the sample; and a flow sensor fluidlyconnected to the flow cell and configured for measuring a sensing signalfrom the sample streams that enter the flow sensor.

In various embodiments, the cartridge device has a size in the range ofabout 0.1-1 cm³, 1-5 cm³, 5-25 cm³, 25-50 cm³, or 50-200 cm³.

In various embodiments, a device as disclosed herein further comprises areader instrument device, wherein the reader instrument device isconfigured for receiving, operating, and/or actuating the cartridgedevice. In various embodiments, the reader instrument device isconfigured for measuring the optical signal at the flow cell to quantifythe target particles in the sample. In various embodiments, the readerinstrument device is configured for measuring the sensing signal at theflow sensor to quantify the volume of the sample streams. In variousembodiments, the reader instrument device is configured for measuringthe optical signal at the flow cell and the sensing signal at the flowsensor to determine the concentration of the target particles in thesample. In various embodiments, the reader instrument device comprises acontrol unit configured for measuring the optical signal at the flowcell. In various embodiments, the reader instrument device comprises acontrol unit configured for measuring the optical signal at the flowcell and the sensing signal at the flow sensor. In various embodiments,the reader instrument device neither receives any liquid from thecartridge device nor transfers any liquid into the cartridge device.

In various embodiments, a cartridge device as disclosed herein furthercomprises a reagent. In various embodiments, the reagent comprises afluorescent labeling agent that selectively labels the target particlesin the sample with fluorescence, and wherein the optical signal from thesample streams comprises fluorescence.

In various embodiments, a cartridge device as disclosed herein furthercomprises a first reagent, which is mixed with a portion of the receivedsample to form a first sample mixture, and a second reagent, which ismixed with another portion of the received sample to form a secondsample mixture; and the two sample mixtures are separately transferredinto the flow cell to form two separate sample streams. In variousembodiments, the two sample mixtures are separately formed in a chamberor separately transferred into a chamber before being separatelytransferred into the flow cell. In various embodiments, the chamber hasa volume in the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml,0.4-0.8 ml, 0.8-2 ml, or 2-10 ml.

In various embodiments, a cartridge device as disclosed herein furthercomprises a fluidic conduit fluidly connected to the inlet andconfigured for receiving or collecting the sample. In variousembodiments, the fluidic conduit is closed by a valve and/or sealed byan external structure after the sample is collected into the fluidicconduit. In accordance with various embodiments of the presentdisclosure, closing by the value and/or sealing by the externalstructure prevents the collected sample from exiting the cartridgedevice. In various embodiments, the fluidic conduit is configured forcollecting a predetermine sample volume in the range of about 0.1-1 μL,1-5 μL, 5-10 μL, 10-20 μL, or 20-50 μL. In various embodiments, at leasta portion of the reagent is transferred into the fluidic conduit toflush a portion of the collected sample into a chamber to form a samplemixture.

In various embodiments, the sample, reagent, sample mixtures, or samplestreams are enclosed inside the cartridge device to prevent or limittheir exposure to the environment outside the cartridge. In variousembodiments, the fluidic structure is inside the cartridge device toprevent or limit exposing the sample, reagent, or sample mixtures to theenvironment outside the cartridge. In various embodiments, the flow cellis inside the cartridge device to prevent or limit exposing the samplestreams to the environment outside the cartridge. In variousembodiments, the flow sensor is inside the cartridge device to preventor limit exposing the sample streams to the environment outside thecartridge.

In various embodiments, the fluidic structure comprises one or aplurality of fluidic conduits. In various embodiments, the fluidicstructure comprises one or a plurality of chambers. In variousembodiments, each chamber has a volume in the range of about 0.01-0.1ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. In certainembodiments, the fluidic structure comprises one or a plurality ofchambers; each chamber has a volume in the range of about 0.01-0.1 ml,0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml; and thefluidic structure is configured for transferring the sample mixturesfrom one of the chambers to the flow cell to form the sample streams.

In some embodiments, a cartridge device as disclosed here comprises oneflow cell. In some embodiments, a cartridge device as disclosedcomprises two, three, four, five, or more flow cells. In someembodiments, a cartridge device as disclosed comprises a plurality offlow cells.

In various embodiments, the flow cell is configured for allowing a flowrate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or200-1000 μl/min. In various embodiments, the flow cell has a crosssection in the shape of a rectangular, trapezoid, oval, circle, or halfcircle, or any other shape, or a combination thereof. In variousembodiments, the flow cell has a width in the range of about 1-10 μm,10-40 μm, 40-100 μm, or 100-200 μm. In various embodiments, the flowcell has a depth in the range of about 1-10 μm, 10-40 μm, 40-100 μm, or100-200 μm. In various embodiments, the flow cell has a length in therange of about of 1-10 μm, 10-100 μm, 100-1,000 μm, 1,000-10,000 μm, or10,000-50,000 μm. In various embodiments, the sample streams formed inthe flow cell have a cross section of the same size as the flow cell.

In certain embodiments, the flow cell has a width in the range of about1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm and a depth in the range ofabout 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm; and the samplestreams have a cross section of the same size as the flow cell.

In various embodiments, the optically transparent area on the flow cellhas a transmission rate of 50-60%, 60-70%, 70-80%, 80-90%, 90-96%, or96-99.9% for the optical signal from the sample streams. In variousembodiments, the optical signal comprises scattered light, reflectedlight, transmitted light, fluorescence, light absorption, lightextinction, or white light image, or a combination thereof. In certainembodiments, the optically transparent area on the flow cell has atransmission rate of 50-60%, 60-70%, 70-80%, 80-90%, 90-96%, or 96-99.9%for the optical signal from the sample streams, and the optical signalcomprises scattered light, reflected light, transmitted light,fluorescence, light absorption, light extinction, or white light image,or a combination thereof.

In various embodiments, the optically transparent area on the flow cellis made of a plastic material. In various embodiments, the plasticmaterial is cyclic olefin copolymer, cyclo-olefin polymer, poly-methylmethacrylate, polycarbonate, polystyrene, orpoly-chloro-tri-fluoro-ethylene, or a combination thereof.

In various embodiments, the flow sensor comprises a fluidic channel anda sensing zone on the fluidic channel; the fluidic channel is fluidlyconnected to the flow cell to allow the sample streams to flow through;and a sensing signal is measured when the sample streams enter thesensing zone. In various embodiments, the sensing signal comprises anoptical signal. In certain embodiments, the optical signal compriseslight transmission through and/or light reflection from the samplestreams.

In various embodiments, the fluidic channel in the flow sensor has achannel width in the range of about 0.001-0.05 mm, 0.05-1 mm, or 1-5 mm,and a channel depth in the range of about 0.001-0.01 mm, 0.01-0.5 mm,0.5-1 mm, or 1-2 mm. In various embodiments, the flow cell and the flowsensor are configured to have the same flow rate for the sample streamsflowing through. In various embodiments, the fluidic connection betweenthe flow cell and the flow sensor is configured for a sample stream tohave the same flow rate flowing through the flow cell and the flowsensor.

In various embodiments, the sensing zone comprises an opticallytransparent area configured for measuring an optical signal that changeslevels between the absence and presence of the sample streams in thesensing zone. In various embodiments, the optically transparent area onthe sensing zone has a transmission rate of 50-60%, 60-70%, 70-80%,80¬90%, 90-96%, or 96-99.9% for the optical signal from the samplestreams. In various embodiments, the optical signal comprises scatteredlight, reflected light, transmitted light, fluorescence, lightabsorption, light extinction, or white light image, or a combinationthereof. In various embodiments, the optically transparent area on thesensing zone is made of a plastic material. In various embodiments, theplastic material is cyclic olefin copolymer, cyclo-olefin polymer,poly-methyl methacrylate, polycarbonate, polystyrene, orpoly-chloro-tri-fluoro-ethylene, or a combination thereof.

In some embodiments, the flow sensor comprises one sensing zone on thefluidic channel. In some embodiments, the flow sensor comprises two,three, four, five, or more sensing zones on the fluidic channel. In someembodiments, the flow sensor comprises a plurality of sensing zones onthe fluidic channel.

In various embodiments, the fluidic structure comprises at least onebasic fluidic unit that comprises: a chamber configured to accommodate afluid; a venting port connected to the chamber, wherein the venting portis connected to a pneumatic pressure source, an ambient pressure, or theatmosphere pressure; a microfluidic channel connected to the chamber;and a valve on the microfluidic channel. In various embodiments, thecartridge device is configured for transferring the sample mixtures fromthe chamber into the flow cell to form the sample streams when anexternal actuation mechanism is applied to the cartridge device.

In various embodiments, the cartridge device is configured fortransferring the sample mixtures from the chamber into the flow cell toform the sample streams when an external actuation mechanism is appliedto the cartridge device. In various embodiments, the external actuationmechanism comprises a pneumatic pressure source. In various embodiments,the external actuation mechanism is configured for forming the samplestreams with a flow rate in the range of 0.001-0.01, 0.01-0.1, 0.1-1,1-50, 50-200, or 200-1000 μl/min. In certain embodiments, the cartridgedevice is configured for transferring the sample mixtures from thechamber into the flow cell to form the sample streams when an externalactuation mechanism is applied to the cartridge device, and the externalactuation mechanism comprises a pneumatic pressure source.

In various embodiments, the chamber of the basic fluidic unit has avolume in the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml,0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. In various embodiments, themicrofluidic channel of the basic fluidic unit has a cross section of asize in the range of about 0.001-0.01 mm², 0.01-0.1 mm², 0.1-0.25 mm²,0.25-0.5 mm², 0.5-1 mm², 1-2 mm², or 2-10 mm². In certain embodiments,the chamber of the basic fluidic unit has a volume in the range of about0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml,and the microfluidic channel of the basic fluidic unit has a crosssection of a size in the range of about 0.001-0.01 mm², 0.01-0.1 mm²,0.1-0.25 mm², 0.25-0.5 mm², 0.5-1 mm², 1-2 mm², or 2-10 mm².

In various embodiments, when the cartridge device is in use, the chamberof the basic fluidic unit is so positioned that the at least a portionof the fluid inside the chamber is pulled by gravity towards themicrofluidic channel and/or away from the venting port. In variousembodiments, when the cartridge device is in use, the chamber of thebasic fluidic unit has a volume larger than the volume of the fluidaccommodated therein and an air gap exists between the venting port andthe fluid accommodated therein.

In various embodiments, the valve of the basic fluidic unit is a passivevalve that is configured for allowing a fluid flow to pass through themicrofluidic channel when a pneumatic pressure is applied to the fluidflow and stopping the fluid flow when no pneumatic pressure is appliedto the fluid flow. In various embodiments, the valve of the basicfluidic unit is a passive valve that comprises one of the followingstructures: (i) a channel with a hydrophilic inner surface embedded witha patch of a hydrophobic surface, (ii) a channel with a hydrophobicinner surface embedded with a patch of a hydrophilic surface, (iii) anenlargement of the channel cross section along the flow direction in achannel with a hydrophilic inner surface, and (iv) a contraction of thechannel cross section along the flow direction in a channel with ahydrophobic inner surface. In various embodiments, the valve of thebasic fluidic unit is an active valve operated by an actuation mechanismexternal to the cartridge device.

Various embodiments of the present disclosure provide a method foranalyzing particles in a sample. The method comprises: providing acartridge device as disclosed herein and a reader instrument device asdisclosed herein; applying the sample to the cartridge device;transferring the cartridge device into the reader instrument device;operating the reader instrument device to actuate the cartridge device;and analyzing the target particles in the sample.

Various embodiments of the present disclosure provide a method foranalyzing target particles in a sample. The method comprises: applyingthe sample to a cartridge device as disclosed herein, which isconfigured for collecting a predetermined sample volume into thecartridge device; transferring the cartridge device into a readerinstrument device as disclosed herein; mixing at least a portion of thecollected sample and at least a portion of a reagent to form one or moresample mixtures inside the cartridge device; forming one or more samplestreams from the one or more sample mixtures in a flow cell inside thecartridge device, wherein the sample streams are formed in the flow cellwithout a sheath flow; measuring an optical signal from the samplestreams at the flow cell to detect the target particles in the samplestreams; and using the reader instrument device to analyze the measuredoptical signal to quantify the target particles in the sample.

Various embodiments of the present disclosure provide a method foranalyzing target particles in a sample. The method comprises: applyingthe sample to a cartridge device as disclosed herein, which isconfigured for collecting a predetermined sample volume into thecartridge device; transferring the cartridge device into a readerinstrument device as disclosed herein; mixing at least a portion of thecollected sample and at least a portion of a reagent to form one or moresample mixtures inside the cartridge device; forming one or more samplestreams from the one or more sample mixtures in a flow cell inside thecartridge device, wherein at least two separate sample mixtures aretransferred into the same flow cell to form at least two separate samplestreams without a sheath flow; measuring an optical signal from thesample streams at the flow cell to detect the target particles in thesample streams; and using the reader instrument device to analyze themeasured optical signal to quantify the target particles in the sample.

In various embodiments, a portion of the collected sample is mixed witha first reagent to form a first sample mixture and another portion ofthe collected sample is mixed with a second reagent to form a secondsample mixture; and the two sample mixtures are separately transferredinto the flow cell to form two separate sample streams. In variousembodiments, the two sample mixtures are separately formed in a chamberor separately transferred into a chamber before being separatelytransferred into the flow cell. In various embodiments, the chamber hasa volume in the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml,0.4-0.8 ml, 0.8-2 ml, or 2-10 ml.

In various embodiments, the sample is collected into a fluidic conduit.In various embodiments, the fluidic conduit is closed by a valve and/orsealed by an external structure after the sample is collected into thefluidic conduit. In accordance with various embodiments of the presentdisclosure, closing by the value and/or sealing by the externalstructure prevents the collected sample from exiting the cartridgedevice. In various embodiments, the fluidic conduit is configured forcollecting a predetermine sample volume in the range of about 0.1-1 μL,1-5 μL, 5-10 μL, 10-20 μL, or 20-50 μL. In various embodiments, at leasta portion of the reagent is transferred into the fluidic conduit toflush a portion of the collected sample into a chamber to form a samplemixture.

In various embodiments, a method as disclosed herein further comprises:flowing the sample streams through a flow sensor that is fluidlyconnected to the flow cell; measuring a sensing signal from the samplestreams at the flow sensor to detect the entrance of the sample streamsinto the flow sensor and/or the exit of the sample streams out of theflow sensor; and using the reader instrument device to analyze themeasured optical signal and sensing signal to determine theconcentration of the target particles in the sample.

In various embodiments, a method as disclosed herein further comprises:flowing the sample streams through a flow sensor that is fluidlyconnected to the flow cell; measuring a sensing signal from the samplestreams at the flow sensor to detect the sample streams entering and/orexiting the flow sensor; and using the reader instrument device toanalyze the measured optical signal and sensing signal to determine theconcentration of the target particles in the sample. In variousembodiments, the sample streams in the flow cell and the flow sensorhave the same flow rate.

In various embodiments, a method as disclosed herein further comprises:flowing the sample streams through a flow sensor that is fluidlyconnected to the flow cell; measuring a sensing signal from the samplestreams at the flow sensor to quantify the volume of the sample streams;and using the reader instrument device to analyze the measured opticalsignal and sensing signal to determine the concentration of the targetparticles in the sample. In various embodiments, the sample streams inthe flow cell and the flow sensor have the same flow rate.

In various embodiments, the optical signal and sensing signal aremeasured by the reader instrument device.

In various embodiments, the collected sample, reagent, sample mixtures,or sample streams are enclosed inside the cartridge device to prevent orlimit their exposure to the environment outside the cartridge.

In various embodiments, the mixing step is performed in a fluidicstructure. In various embodiments, the fluidic structure comprises oneor a plurality of fluidic conduits. In various embodiments, the fluidicstructure comprises one or a plurality of chambers. In variousembodiments, each chamber has a volume in the range of about 0.01-0.1ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. In certainembodiments, the mixing step is performed in a fluidic structurecomprising one or a plurality of chambers, and each chamber has a volumein the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml,0.8-2 ml, or 2-10 ml.

In various embodiments, the fluidic structure is inside the cartridgedevice to prevent or limit exposing the sample, reagent, or samplemixtures to the environment outside the cartridge. In variousembodiments, the flow cell is inside the cartridge device to prevent orlimit exposing the sample streams to the environment outside thecartridge. In various embodiments, the flow sensor is inside thecartridge device to prevent or limit exposing the sample streams to theenvironment outside the cartridge.

In various embodiments, the flow cell has a width in the range of about1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm. In various embodiments, theflow cell has a depth in the range of about 1-10 μm, 10-40 μm, 40-100μm, or 100-200 μm. In various embodiments, the flow cell has a length inthe range of about of 1-10 μm, 10-100 μm, 100-1,000 μm, 1,000-10,000 μm,or 10,000-50,000 μm. In various embodiments, the sample streams formedin the flow cell have a cross section of the same size as the flow cell.

In certain embodiments, the flow cell has a width in the range of about1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm and a depth in the range ofabout 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm; and the samplestreams have a cross section of the same size as the flow cell.

In various embodiments, the sample streams in the flow cell have a flowrate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or200-1000 μl/min when the optical signal is measured from the samplestreams. In various embodiments, the optical signal measured from thesample streams at the flow cell comprises scattered light, reflectedlight, transmitted light, fluorescence, light absorption, lightextinction, or white light image, or a combination thereof.

In various embodiments, the sample streams in the flow sensor have aflow rate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or200-1000 μl/min when the sensing signal is measured from the samplestreams. In various embodiments, the sensing signal measured from thesample streams at the flow sensor comprises an optical signal. Invarious embodiments, the optical signal comprises light transmissionthrough and/or light reflection from the sample streams.

In various embodiments, the sample streams have the same flow rate inthe flow cell and the flow sensor.

In various embodiments, the reagent comprises a fluorescent labelingagent that selectively labels the target particles in the sample withfluorescence, and wherein the optical signal from the sample streamscomprises fluorescence.

In various embodiments, each of the sample streams is separately formedand measured in the flow cell. In various embodiments, at least twoseparate sample mixtures are transferred into the same flow cell to format least two separate sample streams. In some embodiments, the at leasttwo separate sample streams are formed consecutively (i.e., immediatelyone after another). In other embodiments, the at least two separatesample streams are formed nonconsecutively (i.e., not immediately oneafter another). In various embodiments, at least one sample streamcomprises white blood cells as the target particles detected in the flowcell and at least another sample stream comprises red blood cells and/orplatelet cells as the target particles detected in the flow cell.

In various embodiments, the fluidic channel in the flow sensor has achannel width in the range of about 0.001-0.05 mm, 0.05-1 mm, or 1-5 mm,and a channel depth in the range of about 0.001-0.01 mm, 0.01-0.5 mm,0.5-1 mm, or 1-2 mm; and wherein the sample streams in the flow cell andthe flow sensor have the same flow rate.

In certain embodiments, mixing is performed in at least one basicfluidic unit that comprises: a chamber configured to accommodate afluid; a venting port connected to the chamber, wherein the venting portis connected to a pneumatic pressure source, an ambient pressure, or theatmosphere pressure; a microfluidic channel connected to the chamber;and a valve on the microfluidic channel. In various embodiments, thechamber of the basic fluidic unit has a volume in the range of about0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2¬10 ml.In various embodiments, the microfluidic channel of the basic fluidicunit has a cross section of a size in the range of about 0.001-0.01 mm²,0.01-0.1 mm², 0.1-0.25 mm², 0.25-0.5 mm², 0.5-1 mm², 1-2 mm², or 2-10mm².

In certain embodiments, mixing is performed in at least one basicfluidic unit that comprises: a chamber configured to accommodate afluid, wherein the chamber has a volume in the range of about 0.01-0.1ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml; a ventingport connected to the chamber, wherein the venting port is connected toa pneumatic pressure source, an ambient pressure, or the atmospherepressure; a microfluidic channel connected to the chamber, wherein themicrofluidic channel has a cross section of a size in the range of about0.001-0.01 mm², 0.01-0.1 mm², 0.1-0.25 mm², 0.25-0.5 mm², 0.5-1 mm², 1¬2mm², or 2-10 mm²; and a valve on the microfluidic channel.

In various embodiments, the sample mixtures are transferred from thechamber into the flow cell to form the sample streams when an externalactuation mechanism is applied to the cartridge device. In variousembodiments, the external actuation mechanism comprises a pneumaticpressure source. In various embodiments, the external actuationmechanism is configured for forming the sample streams with a flow ratein the range of 0.001¬0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or 200-1000μl/min. In certain embodiments, the sample mixtures are transferred fromthe chamber into the flow cell to form the sample streams when anexternal actuation mechanism is applied to the cartridge device, and theexternal actuation mechanism comprises a pneumatic pressure source.

In various embodiments, the target particles have a size in the range of0.1-1 μm, 1-10 μm, 10-15 μm, 15-30 μm, 30-50 μm, or 50-100 μm. Invarious embodiments, the target particles have a concentration in therange of 1-100, 100-1000, 1000-5000, 5000-20,000, or 20,000-50,000target particles per μl sample steam. In certain embodiments, the targetparticles have a size in the range of 0.1-1 μm, 1-10 μm, 10-15 μm, 15-30μm, 30-50 μm, or 50¬100 μm; and the target particles have aconcentration in the range of 1-100, 100-1000, 1000¬5000, 5000-20,000,or 20,000-50,000 target particles per μl sample steam.

In various embodiments, the target particles comprise cells, plantcells, animal cells, blood cells, white blood cells, red blood cells,platelet cells, viruses, bacteria, fungi, yeasts, beads, fluorescentbeads, or non-fluorescent beads; or other particles of proteins,enzymes, nucleic acids, polysaccharides, or polypeptides; or otherparticles bound with biological markers; or their combinations.

FIG. 1 illustrates one non-limiting example of the basic fluidic unit tobe used in the cartridge. The basic fluidic unit 1001 has a chamber1002, a venting port 1003 and at least one microfluidic channel 1004that accesses the chamber and has a valve 1005 on the microfluidicchannel. The operation of basic fluidic unit 1001 depends on gravity orany other force serving as the replacement for gravity (e.g.,centrifugal force) to keep fluid in position. Additionally, the basicfluidic unit 1001 uses another force such as pneumatic pressure totransfer fluid. More information regarding the design, operation, andmanufacturing of fluid unit 1001 can be found in U.S. application Ser.No. 15/176,729 and PCT Application PCT/US16/36426, which areincorporated herein by reference in their entirety as if fully setforth. In some embodiments, the valve 1005 can be a passive valve. Inother embodiments, the valve 1005 can be an active valve. In certainembodiments, the valve 1005 can be a hybrid or combination of passiveand active valves. In other embodiments, the valve 1005 can be anydesign known to one of ordinary skill in the art.

FIGS. 2A-2D illustrate a few non-limiting examples of passive valves.Other passive valve designs known to persons skilled in the art can alsobe used. FIG. 2A is a passive valve design having a channel with ahydrophilic inner surface and a patch of a hydrophobic surface. FIG. 2Bis a passive valve design having a channel with a hydrophobic innersurface and a patch of a hydrophilic surface. FIG. 2C is a passive valvedesign having an enlargement of the channel cross-section along the flowdirection and the channel has a hydrophilic surface. FIG. 2D is apassive valve design having a narrow down of the channel cross-sectionalong the flow direction and the channel has a hydrophobic surface.

FIGS. 3A-3C illustrate a few non-limiting examples of active valves.Other active valve designs known to persons skilled in the art can alsobe used. FIG. 3A shows a valve design that includes a flexible membrane3001 and a substrate 3002. When the flexible membrane 3001 is bent awayfrom the substrate 3002, the valve is in an “open” status to allow fluidflow to pass through. When the flexible membrane 3001 is bent towardsthe substrate 3002 leaving no gap, the valve is in the “close” statusand fluid flow is not able to pass through. FIG. 3B shows a valve designthat has a movable membrane 3003 and a substrate 3004. When the movablemembrane 3003 is away from the substrate 3004, there is a fluid path3005 between the inlet and outlet, and the valve is in “open” status.When the movable membrane is in proximity with the substrate leaving nogap, there is no fluid path between the inlet 3006 and the outlet 3007,and the valve is in “close” status. FIG. 3C shows a valve design thathas a plug 3008 on the channel. When the plug 3008 is pulled away fromthe channel leaving the substrate 3009, the channel is in the “open”status allowing fluid flow from the inlet 3010 to the outlet 3011. Whenthe plug 3008 is inserted into the channel contacting the substrate3009, the channel is in the “close” status and there is no fluid pathbetween the inlet 3010 and the outlet 3011. The plug 3008 can be made ofsolid material, a polymer, an elastomer, a gel, a wax, a silicon oil, orother materials. When an active valve is used, an additional actuationmechanism can be used to operate the valve.

FIG. 4A illustrates another non-limiting example of implementing apassive valve in a basic fluidic unit 4001. In an embodiment, thetransition area 4005 from the chamber 4002 to the channel 4004 providesa narrowing of the flow channel cross section. When both the channelinner surface and the chamber inner surface within this transition area4005 are hydrophobic, as shown in FIG. 4B, this transition area 4005 isequivalent to the sudden narrow down of a hydrophobic channel and actsas a passive valve to stop fluid in the chamber 4002 from entering thechannel 4004. When both the channel inner surface and the chamber innersurface within this transition area 4005 are hydrophilic, as shown inFIG. 4C, this transition area is equivalent to the sudden enlargement ofa hydrophobic channel and acts as a passive valve to stop fluid in thechannel 4004 from entering the chamber 4002. Additional designs ofpassive valves known to person skilled in the art can also beimplemented.

FIG. 5 illustrates a symbol drawing that represents a basic fluidic unitas described herein, where the basic fluidic unit 5001 includes achamber 5002, a venting port 5003 and at least one microfluidic channel5004 that accesses the chamber and has a valve 5005 on the microfluidicchannel. The valve 5005 can be either a passive valve, an active valveor a hybrid or combination of both. In some embodiments, the basicfluidic unit 5001 can have one or a plurality of microfluidic channels(each having a valve) accessing the chamber 5002 (see, e.g., U.S.application Ser. No. 15/176,729 and PCT Application PCT/US16/36426,which are incorporated herein by reference in their entirety as if fullyset forth).

In various embodiments, present disclosure provides fluidic cartridgeshaving at least one basic fluidic unit as described herein. In variousembodiments, the fluidic cartridges may have additional fluidicstructures. One example of additional fluidic structure is one or moreflow cells for a cytometer analysis. With conventional flow cytometers,the flow cell usually has a core diameter of several hundreds ofmicrometers. To achieve a sample stream of a smaller core diameter,e.g., a few to dozens of micrometers, the flow cell utilizes sheath flowto focus the sample stream. In some embodiments, a fluidic cartridge asdescribed herein includes a conventional flow cell with sheath flow.

In other embodiments, a fluidic cartridge as described herein includes asheathless flow cell instead of the conventional flow cell with sheathflow. The sheathless flow cell has a fluidic channel having a corediameter chosen according to the target sample stream diameter. Forexample, a fluidic channel having a diameter of 30 μm can be used toachieve a target sample stream having a diameter of 30 μm. Additionally,the channel of the flow cell can be transparent to certain excitationlight and emission light wavelengths, so that optical signals can bemeasured from samples in the flow cell (FIG. 6A). FIG. 6B illustrates asymbolic drawing that represents a sheathless flow cell as describedherein. Since the flow cell does not utilize sheath flow, the samplestream has a cross section in the same size as the flow cell.

Another example of an additional fluidic structures is one or aplurality of flow sensors for detecting the sample stream. Describedherein are the design and operation of such a flow sensor, which has oneor a plurality of sensing zones on a channel to detect the existence ofliquid in the channel and/or measure the fluid displacement volume, thevolume of a fluidic plug, flow rate or flow velocity, etc. Moreinformation regarding the design, operation and manufacturing of theflow sensor can be found in U.S. application Ser. No. 15/209,226 and PCTApplication PCT/US16/42089, which are incorporated herein by referencein their entirety as if fully set forth. FIG. 7A illustrates onenon-limiting example of the flow sensor, which has two sensing zonesalong the length of a fluidic channel. The sensor detects whether thereis fluid inside the channel overlapping with the sensing zones. Thevolume of fluid filling up the channel between the two sensing zones canbe determined by the known geometry of the channel. FIG. 7B is asymbolic drawing to represent this design. FIG. 8A illustrates anothernon-limiting example of the flow sensor, which has only one sensing zonealong the length of a fluidic channel. FIG. 8B is a symbolic drawing torepresent this design.

Described herein are various fluidic units and additional fluidicstructures that can be used together in various configurations toachieve functions of a flow cytometer analysis integrating samplepreparation and performing absolute count in a self-contained cartridge.

FIG. 9A shows one exemplary configuration, where a basic fluidic unit9001, a sheathless flow cell 9007 and a flow sensor 9009 with twosensing zones 9011 and 9012 are connected in serial by fluidic conduits9006 and 9008. In some embodiments, the upstream end of the flow cell9007 is connected to the microfluidic channel 9004 of the basic fluidicunit 9001, and the downstream end of the flow cell 9007 is connectedwith the flow sensor. In this particular configuration, sample in thechamber 9002 of the unit 9001 will pass through the flow cell first andthen through the flow sensor for a cytometer analysis.

When using this configuration for a cytometer analysis, as shown in FIG.9B, a fluid sample 9101 can first be loaded into the chamber 9002.Pneumatic pressures can then be applied to the vent 9003 of the basicfluidic unit 9001 and to the outlet port 9010 of the flow sensor. Whenthe pneumatic pressure at vent 9003 is higher than the pneumaticpressure at port 9010, it creates a pressure difference that pumpssample 9101 from the chamber 9002 into the flow cell 9007 for thecytometer analysis, and then into the flow sensor 9009 for volumemeasurement. When the valve 9005 is a passive valve, a sufficiently-highpressure difference can pump the fluid sample to pass the valve 9005.When the valve 9005 is an active valve, the valve 9005 can be switchedto open status before the pressure can pump the fluid sample 9101 topass the valve 9005.

After applying the pneumatic pressures, data of the cytometer analysisin flow cell 9007 is continuously recorded. The recorded data includesthe measured physical signal A (optical emission, electrical impedance,etc.) along time T as an array (A, T). FIG. 9C shows one example of therecorded data, where the amplitude of the signal A is plotted againstthe time T. The number of particles detected in the cytometer isdetermined by the number of peaks in the signal A. Meanwhile, as thesample continues to pass through the flow sensor 9009, the time point T₁of the sample reaching the first sensing zone 9011 is recorded, and thetime point T₂ of the sample reaching the second zone 9012 is alsorecorded. The number of particles N detected between T₁ and T₂ can bedetermined from the record signal (A, T) as shown in the example of FIG.9C. The fluid volume V₀ for filling up the channel between the sensingzone 9011 and the sensing zone 9012 is a known parameter from the flowsensor design (see, e.g., U.S. application Ser. No. 15/209,226 and PCTApplication PCT/US16/42089, which are incorporated herein by referencein their entirety as if fully set forth). Because the sheathless flowcell contains only the fluid sample for analysis (no sheath flow), thefluid volume between the two sensing zones can be used to determine thevolume of sample analyzed in the flow cell 9007. Therefore, the absolutecount can then be calculated as:

Absolute Count=N/V ₀  [1]

In addition to the function of a cytometer analysis with absolute count,the example in FIGS. 9A-9C also has the feature that the whole fluidicstructure can be implemented in a self-contained cartridge device. Thecartridge device can be a molded piece of plastic with additionalsealing layers.

FIG. 10A shows another exemplary configuration, where a flow sensor10007 is connected to the microfluidic channel 10004 of a basic fluidicunit 10001 with a fluidic conduit 10006. Meanwhile, a sheathless flowcell 10009 is connected downstream of the flow sensor 10007 by a fluidicconduit 10008. In this example, the number of cells counted N isdetermined by the signal (A, T) between time points T₁+ΔT and T₂+ΔT, asshown in FIG. 10B, where ΔT can be any empirical value to compensate thetime delay between the sample reaching the first sensing zone 10011 andsample reaching the flow cell 10009. Like the operation of FIG. 9Adiscussed above, the configuration of FIG. 10A can also be used for acytometer analysis with absolute count:

Absolute Count=N/V ₀  [2]

FIG. 11A shows another exemplary configuration, where a basic fluidicunit 11001, a sheathless flow cell 11007 and a flow sensor 11009 withone sensing zone 11012 are connected in serial by fluidic conduits 11006and 11008. In some embodiments, the upstream end of the flow cell isconnected to the microfluidic channel 11004 of the basic fluidic unit11001, and the downstream end of the flow cell is connected to the flowsensor. In this exemplary configuration, sample in the chamber 11002 ofthe unit 11001 passes through the flow cell first and then through theflow sensor for a cytometer analysis. In this example, as shown in FIG.11B, the time points T₁=0 (when the flow cell starts to detectparticles) and T₂ (when the sample reaches the sensing zone 11012) areused to determine the total particle count N from the recorded signal(A, T). Additionally, the fluid volume V₁ to obtain the particle count Nincludes the total fluid conduit volume between the flow sensor 11007and the sensing zone 11012 of the flow sensor 11009. The fluid volume V₁is a parameter known from the fluidic design. Like the operation of FIG.9 discussed above, the configuration of FIG. 10 can also be used forcytometer analysis with absolute count:

Absolute Count=N/V ₁  [3]

FIG. 12A shows another exemplary configuration, where two basic fluidicunits 12101 and 12201 are used in serial with a sheathless flow cell12301 and a flow sensor 12401. A fluidic conduit 12001 connects thebasic fluidic unit 12101's microfluidic channel 12104 (having a valve12105) with the basic fluidic unit 12201's microfluidic channel 12204(having a valve 12205). The unit 12201 has a second microfluidic channel12206 (having a valve 12207), which connects to the upstream end of theflow cell 12301 by a fluid conduit 12002. The downstream end of the flowcell 12301 is further connected to the flow sensor 12401 by a fluidconduit 12003. In this example, the flow sensor 12401 has two sensingzones 12402 and 12403.

For a cytometer analysis, pneumatic pressures are applied to threeports, including the vent 12103 of unit 12101 (P₁), the vent 12203 (P₂)of unit 12201, and at the downstream port 12404 (P₃) of the flow sensor12401. By controlling the applied pneumatic pressures (P₁, P₂, and P₃),a fluid sample can be transferred between the chamber 12102 and thechamber 12202, and further transferred into the flow cell for acytometer analysis with the absolute count.

An exemplary method of controlling the pneumatic pressures (P₁, P₂, andP₃) and the corresponding fluid transfer is shown in the diagram of FIG.12B. When a fluid sample is in the first chamber (chamber 12102), byapplying a pneumatic control (P₁>P₀ and P₂=P₃=P₀), the fluid sample canbe transferred from the first chamber into the second chamber (chamber12202). Similarly, by applying a pneumatic control (P₁<P₀ and P₂=P₃=P₀),the fluid sample can be transferred from the second chamber into thefirst chamber. When the fluid sample is in the second chamber, byapplying a pneumatic control (P₁=P₂=P₀ and P₃<P₀), the fluid sample canbe transferred from the second chamber into the flow cell and the flowsensor for the cytometer analysis. In this exemplary pneumatic controlmethod, the vent port of the second chamber P₂ is kept at a constantpressure P₂=P₀ during the operation. Following the teachings of thisdisclosure and the Applicants' previous disclosures (see, e.g., U.S.application Ser. No. 15/176,729 and PCT Application PCT/US16/36426,which are incorporated herein by reference in their entirety as if fullyset forth), other methods can also be used to control the fluid transferin the configuration. For example, by applying a pneumatic control(P₁=P₂>P₀ and P₃=P₀), the fluid sample in the second chamber can betransferred from the second chamber into the flow cell and the flowsensor for the cytometer analysis. In certain embodiments, P₀ is theatmosphere pressure where the fluidic configuration is operated in.

With the pneumatic control method, a sample can be transferred in thefluidic configuration for the cytometer analysis with absolute count.For example, a fluid sample can be initially introduced into the firstchamber (chamber 12102). The sample can then be transferred to thesecond chamber (chamber 12202). The sample can then be driven throughthe flow cell and the flow sensor for the cytometer analysis withabsolute count as described above. In another example, the fluid samplebe can initially introduced into the second chamber and next driventhrough the flow cell and the flow sensor for the cytometer analysiswith absolute count. In yet another example, a fluid sample A can beinitially introduced into the first chamber and a fluid sample B can beinitially introduced to the second chamber, and then the two samples canbe transferred between the two chambers for a plurality of mixingcycles, before being delivered to the flow cell for the cytometeranalysis with the absolute count. This mixing action involves the fluidsample moving along one direction from the first chamber into the secondchamber and then along an opposite direction from the second chamberinto the first chamber, and vice versa. In some embodiments, the fluidsample A has a predetermined volume. In certain embodiments, the fluidsample B has a predetermined volume.

In another embodiment, a fluid sample A can be initially introduced tothe first chamber 12102 and a fluid sample C can be initially loaded inthe fluid conduit 12001. By transferring the fluid sample between thetwo chambers, the fluid A and the fluid C can be mixed together, andthen delivered to the flow cell for the cytometer analysis with theabsolute count. The sample exiting the outlet of the flow sensor can bedisposed or collected in a reservoir. In an embodiment where a reservoiris used to collect the exiting sample, the fluidic configuration of thisexample achieves the sample preparation, cytometer analysis and theabsolute count function in a self-contained manner, without an exchangeof fluid sample between the fluidic structure and the outsideenvironment. Such an embodiment can be used for a cytometer analysis ofdifferent biological samples. For example, the fluid sample C can be abiological sample such as whole blood from human body. The fluid sampleA can be a reagent containing a fluorophore-conjugated antibodytargeting specific cell types, e.g., CD4+ lymphocytes, in the wholeblood. By mixing these two fluids together and then measuring themixture in the flow cell, it achieves the absolute count of the CD4+Lymphocyte. In another example, the fluid sample C can be a human wholeblood, whereas the fluid sample A can be a lysing solution selectivelytargeting Red Blood Cells (RBCs). After mixing the two fluids togetherand incubating the mixture for a period of time, the mixture is measuredin the flow cell for a cytometer analysis such as the absolute count ofthe white blood cells (WBCs).

FIG. 12C shows an exemplary fluidic configuration, where an additionalfluid conduit 12004 is connected to the fluid conduit 12001 to introducethe initial sample C. The sample can be introduced via the port 12006into the fluidic conduit 12001. A valve 12005 can then be closed to sealthe conduit 12004, preventing the sample from exiting the port 12006. Insome embodiments, the fluid conduit 12001 can be used to collect apredetermined volume of the initial sample. In some embodiments, thevalve 12005 can be a blood clotting valve (see, e.g., U.S. Pat. No.8,845,979, which incorporated herein by reference in its entirety as iffully set forth). Other methods known to persons skilled in the art canalso be used to introduce the initial samples.

In some embodiments, a reservoir chamber can be connected to outlet portof the flow sensor to collect the fluid sample after the cytometeranalysis. As shown in the exemplary fluidic configuration of FIG. 12D, areservoir 12501 with a venting port 12502 can be connected to the outletport 12404 of the flow sensor by a fluidic conduit 12503. With thisextra reservoir, the pneumatic pressure P₃ can be adjusted bycontrolling the pneumatic pressure P₄ at the venting port 12502 of thereservoir. An exemplary operation of this fluidic configuration isillustrated in FIG. 12E.

In other embodiments, additional fluidic structures can be connected toone or more of the basic fluidic units, achieving additional operationfunctionalities. FIG. 12F shows one example of these embodiments. Incomparison to the example of FIG. 12A, the second basic fluidic unit12201 has a third microfluidic channel 12208 (with a valve 12209), whichconnects by a fluidic conduit 12007 to a reservoir structure 12601 witha venting port 12602. The venting port 12602 corresponds to a pneumaticpressure P₅. A fluid sample D is initially stored in the reservoir12601. FIG. 12G shows a pneumatic control for operating thisconfiguration. By applying a pneumatic control (P₅>P₀ and P₂=P₀), thesample D initially stored in the reservoir 12601 is transferred into thesecond chamber 12202. The rest of the pneumatic operation for thecytometer analysis can be the same as the example in the FIG. 12B.

In yet another exemplary configuration, as shown in FIG. 13A, there arethree basic fluidic units 13101, 13201 and 13301. The units 13101 and13201 have microfluidic channels 13104 (with the valve 13105) and 13204(with the valve 13205), respectively. The unit 13301 has threemicrofluidic channels 13304 (with the valve 13305), 13306 (with thevalve 13307) and 13308 (with the valve 13309). A fluidic conduit 13001connects the channels 13104 and 13304, whereas another fluidic conduit13002 connects the channel 13204 and 13306. The fluid unit 13302 is alsoconnected to an upstream port of a sheathless flow cell 13401 by afluidic conduit 13003, while the downstream port of the flow cell 13401is connected to a flow sensor 13501 by a fluidic conduit 13004. Thisfluidic configuration is operated by controlling the pneumatic pressuresat the venting ports of the basic fluidic units 13103 (P₁), 13203 (P₂)and 13303 (P₃), and by controlling the pneumatic pressure of the outletport 13504 of the flow sensor (P₄).

An exemplary method of controlling the pneumatic pressures (P₁, P₂, P₃and P₄) and the corresponding fluid transfer is shown in the diagram ofFIG. 13B. A fluid sample in the first chamber (13102) can be transferredinto the third chamber (13302) by applying a pneumatic control (P₁>P₀and P₂=P₃=P₄=P₀), whereas a fluid sample in the third chamber can betransferred into the first chamber by applying a pneumatic control(P₁<P₀ and P₂=P₃=P₄=P₀). Similarly, a fluid sample in the second chamber(13202) can be transferred into the third chamber by applying apneumatic control (P₂>P₀ and P₁=P₃=P₄=P₀), whereas a fluid sample in thethird chamber can be transferred into the second chamber by applying apneumatic control (P₂<P₀ and P₁=P₃=P₄=P₀). Meanwhile, a fluid sample inthe third chamber can be transferred into the flow cell and the flowsensor for the cytometer analysis, by applying a pneumatic control(P₄<P₀ and P₁=P₂=P₃=P₀). In this exemplary pneumatic control method, thevent port of the third chamber P₃ is kept at a constant pressure P₃=P₀during the operations. Following the teachings of this disclosure andApplicants' previous disclosures (see, e.g., U.S. application Ser. No.15/176,729 and PCT Application PCT/US16/36426, which are incorporatedherein by reference in their entirety as if fully set forth), othermethods can also be used to control the fluid transfer in thisconfiguration.

This fluidic configuration and the fluid transfer diagram can be used toperform more complex sample preparation and cytometer analysis. Forexample, as shown in FIG. 13C, a fluid sample A1 is initially introducedinto the first chamber and a fluid sample A2 is initially introducedinto the second chamber. Meanwhile, fluid samples B1 and B2 are eachintroduced into the fluidic conduit 13001 and 13002, respectively. Byapplying the pneumatic control (P₁>P₀ and P₂=P₃=P₄=P₀), the fluidsamples A1 and B1 are transferred into the third chamber. By applyingthe pneumatic control (P₄<P₀ and P₁=P₂=P₃=P₀), the sample mixture of A1and B1 is transferred into the sheathless flow cell and the flow sensorfor the cytometer analysis with the absolute count. After the cytometeranalysis, if any residue of the sample mixture is left behind in thethird chamber, it can be transferred back into the first chamber byapplying pneumatic control (P₁<P₀ and P₂=P₃=P₄=P₀) and thus make thethird chamber empty to be ready for analysis of other samples. Prior tothe cytometer analysis, if mixing uniformity of the sample mixture is adesign consideration, the pneumatic control (P₁<P₀ and P₂=P₃=P₄=P₀) and(P₁>P₀ and P₂=P₃=P₄=P₀) can be applied in sequential to move the mixturefrom the third chamber into the first chamber, and then from the firstchamber back into the third chamber again, introducing a mixing actionof the sample. This step can be repeated to achieve desirable mixinguniformity. During these steps of fluid transfer, the fluid samples A2and B2 do not move. This is achieved by keeping the pneumatic pressure(P₂=P₃). Next, the fluid samples A2 and B2 can be transferred into thethird chamber by applying the pneumatic control (P₂>P₀ and P₁=P₃=P₄=P₀).The mixture of the samples A2 and B2 can be next transferred into theflow cell for the cytometer by applying the pneumatic control (P₄<P₀ andP₁=P₂=P₃=P₀). If mixing uniformity is desirable, steps of repeatedtransfer between the second chamber and the third chamber can also becarried out similar to the repeated transfer steps between the first andthe third chamber. In certain embodiments, the fluid sample A1 has apredetermined volume. In certain embodiments, the fluid sample A2 haspredetermined volume. In certain embodiments, the fluid sample B1 haspredetermined volume. In certain embodiments, the fluid sample B2 haspredetermined volume.

This fluid configuration and the fluid transfer diagram can be used toimplement a cytometer analysis of various biological samples. Forexample, the fluid sample A1 can be a dilution buffer for RBC analysisand the sample B1 can be whole blood, whereas the sample A2 can be alysing buffer for WBC analysis and the sample B2 can be whole blood. A1and B1 can be transferred into the third chamber to form a mixture, andthen into the flow cell for counting and analyzing of RBCs and plateletsin the blood. A2 and B2 can then be transferred into the third chamberto form a mixture, and then into the flow cell for counting andanalyzing WBCs in the blood. Different dilution buffers and lysingbuffers known to persons skilled in the art of hematology analyzers canbe used. In this way, the fluid configuration can be used to achieve theComplete Blood Count (CBC) analysis widely used in clinical tests.

FIGS. 12A-12G and FIGS. 13A-13C show examples having two and three basicfluidic units, respectively. In other embodiments, more basic fluidicunits can be used in the configuration to achieve additional complexity.In the examples of FIGS. 12A-12G and FIGS. 13A-13C, the fluidic conduitsfor connecting the basic fluidic units (e.g., the fluid conduit 12001,the fluid conduit 13001 and the fluid conduit 13002) are fluid channels.In other embodiments, fluid structures with additional complexity can beused as the fluidic conduits.

FIG. 14A shows an example with four basic fluidic units, 14101, 14201,14301 and 14401. Each of the four units 14010, 14201 and 14401 has amicrofluidic channel with a valve. The unit 14301 has four microfluidicchannels including channel 14304 (with valve 14305), channel 14306 (withvalve 14307), channel 14308 (with valve 14309), and channel 14310 (withvalve 14311). The basic fluidic unit 14101 is connected to the basicfluidic unit 14301 by a fluidic conduit 14001. The basic fluidic unit14201 is connected to the basic fluidic unit 14301 by a fluidic conduit14002. The basic fluidic unit 14401 is connected to the basic fluidicunit 14301 by a fluidic conduit 14003. The upstream of a sheathless flowcell 14501 is connected to the basic fluidic unit 14301 by a fluidicconduit 14004, while the downstream of the flow cell 14501 is connectedby a fluidic conduit 14005 to a flow sensor 14601 that has two sensingzones 14602 and 14603. The flow sensor 14601 is then connected to areservoir chamber 14701 that has a venting port 14702. Meanwhile, asample A1 can be initially stored in the chamber 14102 of the basicfluidic unit 14101, a sample A2 can be initially stored in the chamber14202 of the basic fluidic unit 14201 and a sample A3 can be initiallystored in the chamber 14402 of the basic fluidic unit 14401.Additionally, a sample B1 can be induced into the fluidic conduit 14001by an inlet port 14801 through a fluidic conduit 14801 with a valve14803. The valve 14803 can be closed after inducing the sample. In someembodiments, the fluidic conduit 14001 can be used to collect apredetermined volume of the sample. Similarly, a sample B2 can beinduced into the fluidic conduit 14002 by an inlet port 14901 through afluidic conduit 14902 with a valve 14903. The valve 14903 can be closedafter inducing the sample. In some embodiments, the fluidic conduit14002 can be used to collect a predetermined volume of the sample.

An exemplary method of controlling the pneumatic pressures (P₁, P₂, P₃,P₄ and P₅) is described below and the corresponding fluid transfer isshown in the diagram of FIG. 14B. The pneumatic pressure P₅ at theventing port 14702 of the reservoir 14701 balances with the pressure P₅′at the downstream port 14604 of the flow sensor 14601, when there is apneumatic path between these two ports (e.g., when there is air path inthe reservoir 14701 to balance the venting port 14702 and the port14604). A fluid sample in the first chamber (14102) can be transferredinto the third chamber (14302) by applying a pneumatic control (P₁>P₀and P₂=P₃=P₄=P₅=P₀), whereas a fluid sample in the third chamber can betransferred into the first chamber by applying a pneumatic control(P₁>P₀ and P₂=P₃=P₄=P₅=P₀ where P₀ is the atmosphere pressure. A fluidsample in the second chamber (14202) can be transferred into the thirdchamber by applying a pneumatic control (P₂>P₀ and P₁=P₃=P₄=P₅=P₀),whereas a fluid sample in the third chamber can be transferred into thesecond chamber by applying a pneumatic control (P₂<P₀ andP₁=P₃=P₄=P₅=P₀). Similarly, a fluid sample in the fourth chamber (14402)can be transferred into the third chamber by applying a pneumaticcontrol (P₄>P₀ and P₁=P₂=P₃=P₅=P₀), whereas a fluid sample in the thirdchamber can be transferred into the fourth chamber by applying apneumatic control (P₄<P₀ and P₁=P₂=P₃=P₅=P₀). Meanwhile, a fluid samplein the third chamber can be transferred into the flow cell and the flowsensor for the cytometer analysis, by applying a pneumatic control(P₅<P₀ and P₁=P₂=P₃=P₄=P₀). In this exemplary pneumatic control method,the vent port of the third chamber P₃ is kept at a constant pressureP₃=P₀ during the operations. Following the teachings of this disclosureand Applicants' previous disclosures (see, e.g., U.S. application Ser.No. 15/176,729 and PCT Application PCT/US16/36426, which areincorporated herein by reference in their entirety as if fully setforth), other methods can also be used to control the fluid transfer inthis configuration.

In various embodiments, the sheathless flow cell is where the targetparticles in a fluid sample flow are detected and measured by differentsignals such as fluorescence, light scattering, light absorption, andlight extinction, white light imaging, etc. An excitation light (EL)beam from a light source can be shaped and used to illuminate adesignated sensing area of the flow cell and trigger the above signalsfrom the target particles.

The sheathless flow cell can be a fluidic channel that has variousgeometry shapes. FIGS. 15A-15D show the top view (in x-y plane) of a fewexamples of the flow cell. The top view (x-y plane) is defined as theplane perpendicular to the direction of the excitation light (z-axis).The length is defined the as channel dimension along the sample flow(x-axis), and the width is defined as the dimension along the y-axis.The depth is defined as the channel dimension along the z-axis. FIG. 15Bshows an example of a flow cell that has a gradually decreased width,where the maximum width is W₁ and the minimum width is W₂. In otherembodiments, the flow cell can have a gradually increased width. FIG.15C shows an example of a flow cell that has a non-gradually changingwidth, where the maximum width is W₁ and the minimum width is W₂. FIG.15D shows an example of a flow cell that has a fixed width (W₁=W₂=W₀) atvarious positions along channel length. In some embodiments, thedifference of the maximum width W₁ and the minimum width W₂ are within adesignated difference. A non-limiting example of the range of the widthdifference is (W₁−W₂)/W₂≤20%. The ranges of the channel width and thedepth are chosen to be large enough so that target particles (e.g.,cells in biological samples), can pass through the flow cell withoutblocking it. Meanwhile, they are chosen to be small enough to minimizethe coincidence error in the flow cytometer analysis. The minimum widthW₁ can be in the range of 1-10 μm, 10-40 μm, 40 to 100 μm, or 100 to 200μm. The depth of the channel can be in the range of 1-10 μm, 10-40 μm,40 to 100 μm, or 100 to 200 μm. The cross section of the channel (in y-zplane) can have the shape of a rectangular, a trapezoid, a circle, ahalf circle, or any other shapes. The length of the flow cell should belong enough for the optical detection of particles in the sample stream,and meanwhile, short enough to reduce the flow resistance of the samplestream flowing through. In various embodiments, the length of the flowcell can be in the range of about 1-10 μm, 10-100 μm, 100-1,000 μm,1,000-10,000 μm, or 10,000-50,000 μm.

As the sheathless flow cell is used for optical measurement, at leastone surface of the channel is transparent to the light wavelengthinvolved in the measurement. The material for forming the channelsurface can be any transparent material such as glass, quartz, andplastics including, but not limited to, Cyclic Olefin Copolymer (COC),Cyclo-olefin Polymer (COP), Poly-Methyl methacrylate (PMMA),polycarbonate (PC), Polystyrene (PS), andPoly-chloro-tri-fluoro-ethylene (PCTFE) materials such as Aclar, etc.

The fluid sample for analysis in the flow cell can be a fluid suspensionof a plurality of particles. For example, the fluid sample can be ablood sample containing different cells such as white blood cells, redblood cells and platelets. In another example, the fluidic sample can bea blood sample, in which certain types of cells remain intact, such aswhite blood cells, while other types of cells have been lysed, such asred blood cells. In another example, the fluid sample can be a bloodsample in which certain types of cells have been labeled with afluorophore. In another example, the fluidic sample can be a mixture ofthe cells and other particles, such as non-fluorescent beads and/orfluorescent beads. In other examples, the fluid samples can also beother biological samples such as cerebrospinal fluid, urine, saliva,semen, etc.

When particles flow through the sheathless flow cell, various opticalsignals can be measured to detect and characterize the particles. Themeasurable signals include, but are not limited to, fluorescence, lightscattering, light absorption, light distinction, etc. FIG. 16A shows anexample where a plurality of particles flow through the flow cell fordetection. All particles in the sample flow through in a one-by-onemanner. Under the illumination of the excitation light (EL), each cellcan be characterized for optical signals that include but are notlimited to fluorescence (FL) and light scattering (LS). FIG. 16B showsanother example where a plurality of particles flow through the flowcell for detection. Some particles are flowing through while overlappingwith each other. Nevertheless, if only considering the target particles,they are flowing through still in a one-by-one manner withoutoverlapping with other target particles. In this case, the lightscattering from the target particles may be blocked by other particlesoverlapping with them. Nevertheless, other signals that include but arenot limited to fluorescence signal can still be measured to detect thesetarget particles if these particles are treated with specificfluorophore to distinguish from the other particles beforehand. In someembodiments, the other particles can be non-fluorescent or treated withdifferent fluorophore distinguishable from the target particles.

In an embodiment, the fluid sample can be a blood sample in which thewhite blood cells are labeled with fluorophore and the red blood cellsare not labeled with fluorophore. When the labeled white blood cellspass through the flow cell one-by-one, the corresponding fluorescencesignal can be measured to detect and characterize the white blood celleven when there are red blood cells overlapping with them. In anotherembodiment, the fluid sample can be a blood sample in which the whiteblood cells are labeled with fluorophore and the red blood cells arelysed. When the labeled white blood cells pass through the flow cellone-by-one, the corresponding fluorescence signal and light scatteringsignals can be measured simultaneously from these cells for detectionand characterization. In another embodiment, the fluid sample can be ablood sample in which there are fluorophore-labeled white blood cellsand fluorescent beads. When these white blood cells and the beads passthrough the flow cell one-by-one, they can be detected by thecorresponding fluorescence signal. Other cells having no fluorescence,or a different wavelength of fluorescence do not impede the measurement.In another embodiment, the fluid sample can be a blood sample in whichthere are red blood cells and beads among other cells. When the cellspass through one-by-one, light scattering signals can be measured todetect and characterize the red blood cells and the beads. In yet otherembodiment, the bead can be label with a fluorophore, so they can bedistinguished from the red blood cells by the light scattering signalsor by fluorescence signals, or by both signals.

A combination of the sheathless flow cell and the flow sensor achievesthe desired functionality of the cytometer analysis with the absolutecount of particles. FIG. 17A shows one exemplary design, where theoutlet 17103 of the flow cell 17101 is coupled to the inlet 17202 of theflow sensor 17201 by a fluidic conduit 17001. In certain embodiments,the outlet of the flow cell 17101 can be coupled to the inlet 17202 ofthe flow sensor 17201 directly and without additional fluidic conduit.The flow sensor 17201 has two sensing zones 17204 and 17205. A fluidsample flows into the inlet 17102 of the flow cell 17101 and then out ofthe outlet 17203 of the flow sensor 17201. The signal measured in theflow cell 17101 is recorded, as illustrated in FIG. 17B. Time T₀ is whenthe sample starts being detected in the flow cell 17101, T₁ is when thefluid sample passes the sensing zone 17204, and T₂ is when the fluidsample passes the sensing zone 17205. From time T₁ to T₂, the totalnumber of target particles detected in the flow cell 17101 is N. Thefluid volume V₀ between the two sensing zones 17204, 17205 is a knownparameter from the design of the flow sensor. Because the flow cell17101 has a sheathless design, the volume of fluid flows through theflow cell 17101 is only the fluid sample. Therefore, the sample volumebeing measured in the flow cell 17101 between T₁ and T₂ equals to V₀. Inthis design, the absolute count is determined as:

Absolute Count 1=N/V ₀  [4]

FIG. 17C shows another exemplary design, where the flow sensor 17201 hasonly one sensing zone 17205. FIG. 17D is the signal measured from thisdesign, where time T₀ is when the sample starts being detected in theflow cell, and T₂ is when the fluid sample passes the sensing zone17205. From time T₀ to T₂, the total number of target particles detectedin the flow cell is N′. The volume V₀′ is the total fluid volume fillingup the fluidic conduit from the flow cell 17101 to the sensing zone17205. In this design, the absolute count is determined as:

Absolute Count 2=N′/V ₀′  [5]

FIG. 18A shows another exemplary design, where the inlet 18102 of theflow cell 18101 is coupled to the outlet 18203 of the flow sensor 18201by a fluidic conduit 18001. The flow sensor 18201 has two sensing zones18204 and 18205. A fluid sample flows into the inlet 18102 of the flowsensor 18201 and then out of the outlet 18203 of the flow sensor 18201.The signal measured in the flow cell 18101 is recorded, as illustratedin FIG. 18B. T₁ is when the fluid sample passes the sensing zone 18204,and T₂ is when the fluid sample passes the sensing zone 18205. In thisexample, the number of cells counted N″ is determined by the signal (A,T) between time points T₁+ΔT and T₂+ΔT, as shown in FIG. 18B, where ΔTcan be any empirical value to compensate the time delay between samplereaching the first sensing zone 18204 and sample reaching the flow cell18101. From time T₁+ΔT to T₂+ΔT, the total number of target particlesdetected in the flow cell is N″. The fluid volume V₀ between the twosensing zones 18204, 18205 is a known parameter from the design of theflow sensor 18201. In this design, the absolute count is determined as:

Absolute Count 3=N″/V ₀  [6]

This combination of the flow cell 18101 and the flow sensor 18201 can beused for measurement of particles or cells. The size of the targetparticles can be in the range of 0.1-1 μm, 1-10 μm, 10-15 μm, 15-30 μm,30-50 μm, or 50-100 μm depending on the size of the flow cell 18101. Tominimize the risk of clogging the sheathless channel, the size of theparticles being measured should be smaller than size of the flow cell18101, and the size difference can range from 1-5 μm, 5-10 μm, 10-20 μm,or 20-50 μm. To minimize the coincidence error for the cytometeranalysis, the concentration of the target particles in the fluid samplecan be in the range of 1-100, 100-1000, 1000-5000, 5000-20,000, or20,000-50,000 particles or cells per μl sample.

When the target particles are biological cells, too fast of a flowvelocity in the sheathless flow cell can introduce shear force and maylyse the cells. Because the sheathless flow cell has a dimension similarto the target particles, this imposes a limitation on the flow rate ofthe sample. The flow rate can be in the range of 0.001-1, 1-50, 50-200,or 200-1000 microliters per minute (μ1/min). In certain embodiments, theranges can be 1-50 or 50-200 μl/min. For size consideration whenimplementing in self-contained cartridges, the range of the fluid samplevolume can be constrained by the cartridge size. The volume of the flowsensor and the total volume of the sample can be in the range of 0.1-1μl, 1-200 μl, 200-1000 μl, 1-5 ml, or 5-30 ml. In certain embodiments,the range can be 1-200 μl, 200-1000 μl l or 1-5 ml. In certainembodiments, by considering both the sample volume and the flow rate,the measurement is completed in less than 10 minutes.

By further incorporating the combination of the sheathless flow cell andthe flow sensor with the basic fluidic unit, as illustrated in theexamples shown in FIGS. 9A-14B, sample preparation steps can be furtherintegrated with the cytometer analysis including the absolute count. Theintegration of the above functions enables the fluidic configurations tobe operated as a self-contained structure for a cytometer analysis,without fluid exchange with the outside environment after the fluidsamples having been loaded into the cartridge.

In different embodiments of the fluidic configurations, pneumaticpressures are applied to the venting ports of the basic fluidic unitsand additional venting ports of other fluidic structures such as areservoir (see, e.g., FIG. 12D and FIG. 14A). The higher the pressuredifference between two venting ports is, the higher the flow rate totransfer fluid in the microfluidic channels. When the fluid sample is abiological sample containing cells, a high flow rate in a confinedchannel may induce a large shear force to lyse the cells. Consideringthis limitation, the pressure difference between any two of the appliedpressures can be in the range of 0-1, 1-5, 5-15, or 15-30 psi. Incertain embodiments, the range can be 0-1, 1-5, or 5-15 psi. In certainembodiments, at least one of the venting ports can be connected to theatmosphere pressure of the environment. When at least one venting portis connected to the atmosphere pressure, another pressure higher thanthe atmosphere pressure applied introduces a positive pressuredifference in comparison to the atmosphere pressure. This positivepressure difference can be in the range of 0-1, 1-5, 5-15, or 15-30 psi.In certain embodiments, the range can be 0-1, 1-5, or 5-15 psi. When atleast one venting port is connected to the atmosphere pressure, anotherpressure lower than the atmosphere pressure applied introduces anegative pressure difference. This negative pressure difference can bein a range of 0-1, 1-5, 5-15, or 15-30 psi. In certain embodiments, therange can be 0-1, 1-5, or 5-15 psi. The flow rate achieved fortransferring the sample via the channel between any two of the basicfluidic units can be in the range of 0-1, 1-50, 50-200, or 200-1000μl/min, or 1-10 ml/min. In certain embodiments, the range can be 1microliter to 1-50, 50-200, or 200-1000 μl/min.

Various fluidic configurations incorporating a plurality of basicfluidic units and a plurality of the combinations of the sheathless flowcell and the flow sensor can be implemented in various manufacturingstructures to form a fluidic cartridge. In some embodiments, thiscartridge can be inserted into a reader instrument for operation, asshown in the example of FIG. 19. The cartridge 19101 having the fluidicstructure 19102 can be inserted into a docking slot 19202 on the readerinstrument 19201. In some embodiments, a control unit of the readerinstrument records the signals from the cytometer analysis. Someexamples of the signals include but are not limited to the opticalsignals such as fluorescence, light scattering, light absorption, etc.In some embodiments, the reader instrument has alignment mechanisms andfeatures to align the sheathless flow cell with the optics in theinstrument for optical signal measurement. In some embodiments, thecontrol unit of the reader instrument also detects the signals from theflow sensor to determine the absolute count. In some embodiments, thecontrol unit of the reader instrument also applies the pneumaticpressure source to the cartridges to drive the fluid transfer. In someembodiments, the control unit of the reader instrument also supportsadditional actuations such as opening or closing a valve structure inthe cartridge fluidics. In some embodiments, the cartridge isself-contained and there is no exchange of liquid samples between thecartridge and the reader instrument. In some embodiments, the cartridgeis not self-contained, and the reader instrument has on-board liquidstorage and there is liquid exchange between the reader instrument andthe cartridge, such as liquid infusion from the reader instrument intothe cartridge.

In some embodiments, the cartridge stays stationary after being insertedinto the reader, whereas the interface for external connections such asthe pneumatic pressure source moves to make contact with the cartridge.In other embodiments, the cartridge can be movable after being insertedinto the reader and is moved to make contact with the interface forexternal connections such as pneumatic pressure sources.

The sheathless flow cell in the fluidic structures can be built withvarious manufacturing processes. In some embodiments, an open fluidicchannel for the flow cell can be built by injection molding, embossing,etching, CNC, laser cutting, or die cutting, etc. A cover can then beadded onto the patterns to form enclosed fluidic channel to be the flowcell. The cover can be added by various manufacturing process, such asthermal fusion bonding, thermal lamination, adhesive bonding, solventassisted bonding, laser wielding, and ultrasonic wielding, etc.Non-limiting examples of building the sheathless flow cell are describedhere. In some embodiments, optical signals are detected from particlesflowing inside the sheathless flow cell. Smooth surface of the flow cellis useful to achieve acceptable optical signals. FIG. 20A shows oneexample of the sheathless flow cell 20101 having two pieces. Thecross-section view (y-z plane) is perpendicular to the direction ofsample flow (x-axis). The bottom piece 20102 forms three sides of achannel without a cover. The top piece 20103 adds a cover side to thechannel, which then forms an enclosed channel. The bottom and the topsurfaces 20104 and 20105 can achieve smoothness for optical measurementin the two pieces 20102 and 20103, respectively. FIG. 20B shows anotherexample of building the sheathless flow cell 20201 having three pieces.The middle piece 20202 forms two sides of a channel, without top andbottom sides. Then a bottom piece 20203 and a top piece 20204 are addedseparately. The three pieces together forms an enclosed channel as theflow cell. The surface 20205 and 20206 can achieve smoothness foroptical measurement in the two pieces 20203 and 20204, respectively.

The cartridge device for the cytometer analysis can be of any size. Incertain embodiment, the cartridge device is received in the readerinstrument device for the measurement and analysis and has a size in therange of about 0.1-1 cm³, 1-5 cm³, 5-25 cm³, 25¬50 cm³, or 50-200 cm³.

Many variations and alternative elements have been disclosed inembodiments of the present disclosure. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of fluidicunits, components and structures for the inventive devices and methods,and the samples that may be analyzed therewith. Various embodiments ofthe disclosure can specifically include or exclude any of thesevariations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the disclosure are tobe understood as being modified in some instances by the term “about.”As one non-limiting example, one of ordinary skill in the art wouldgenerally consider a value difference (increase or decrease) no morethan 10% to be in the meaning of the term “about.” Accordingly, in someembodiments, the numerical parameters set forth in the writtendescription and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of thedisclosure may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The disclosure is explained by various examples, which are intended tobe purely exemplary of the disclosure and should not be considered aslimiting the disclosure in any way. Various examples are provided tobetter illustrate the claimed disclosure and are not to be interpretedas limiting the scope of the disclosure. To the extent that specificmaterials are mentioned, it is merely for purposes of illustration andis not intended to limit the disclosure. One skilled in the art maydevelop equivalent means or reactants without the exercise of inventivecapacity and without departing from the scope of the disclosure.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature, or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specificallyincluded, and others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

Various embodiments of the disclosure are described above in theDetailed Description. While these descriptions directly describe theabove embodiments, it is understood that those skilled in the art mayconceive modifications and/or variations to the specific embodimentsshown and described herein. Any such modifications or variations thatfall within the purview of this description are intended to be includedtherein as well. Unless specifically noted, it is the intention of theinventors that the words and phrases in the specification and claims begiven the ordinary and accustomed meanings to those of ordinary skill inthe applicable art(s).

The foregoing description of various embodiments of the disclosure knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the disclosure to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe disclosure and its practical application and to enable othersskilled in the art to utilize the disclosure in various embodiments andwith various modifications as are suited to the particular usecontemplated. Therefore, it is intended that the disclosure not belimited to the particular embodiments disclosed for carrying out thedisclosure.

While particular embodiments of the present disclosure have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this disclosure and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this disclosure.

Additional Aspects of the Present Disclosure

Aspects of the subject matter described herein may be useful alone or incombination with any one or more of the other aspect described herein.Without limiting the foregoing description, in a first aspect of thepresent disclosure, a device for analyzing target particles in a sampleincludes a cartridge device, wherein the cartridge device comprises: aninlet configured for receiving the sample into the cartridge device; afluidic structure fluidly connected to the inlet and configured formixing at least a portion of the sample with at least a portion of areagent to form one or more sample mixtures; a flow cell fluidlyconnected to the fluidic structure and configured for forming one ormore sample streams from the one or more sample mixtures, wherein thesample streams are formed in the flow cell without a sheath flow, andwherein the flow cell comprises an optically transparent area configuredfor measuring an optical signal from the sample streams to detect thetarget particles in the sample; and a flow sensor fluidly connected tothe flow cell and configured for measuring a sensing signal from thesample streams that enter the flow sensor.

In accordance with a second aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the fluidic structure comprises one or a plurality ofchambers, wherein each chamber has a volume in the range of about0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml;and wherein the fluidic structure is configured for transferring thesample mixtures from one of the chambers to the flow cell to form thesample streams.

In accordance with a third aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the flow cell has a width in the range of about 1-10 μm,10-40 μm, 40-100 μm, or 100-200 μm and a depth in the range of about1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm; and wherein the samplestreams have a cross section of the same size as the flow cell.

In accordance with a fourth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the flow cell has a length in the range of about of 1-10μm, 10-100 μm, 100-1,000 μm, 1,000-10,000 μm, or 10,000-50,000 μm.

In accordance with a fifth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the optically transparent area on the flow cell has atransmission rate of 50-60%, 60-70%, 70-80%, 80-90%, 90-96%, or 96-99.9%for the optical signal from the sample streams, and wherein the opticalsignal comprises scattered light, reflected light, transmitted light,fluorescence, light absorption, light extinction, or white light image,or a combination thereof.

In accordance with a sixth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the optically transparent area on the flow cell is madeof a plastic material.

In accordance with a seventh aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the cartridge device further comprises a reagent.

In accordance with an eighth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the reagent comprises a fluorescent labeling agent thatselectively labels the target particles in the sample with fluorescence,and wherein the optical signal from the sample streams comprisesfluorescence.

In accordance with a ninth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the flow sensor comprises a fluidic channel and a sensingzone on the fluidic channel, wherein the fluidic channel is fluidlyconnected to the flow cell to allow the sample streams to flow through;and wherein a sensing signal is measured when the sample streams enterthe sensing zone.

In accordance with a tenth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the fluidic channel in the flow sensor has a channelwidth in the range of about 0.001-0.05 mm, 0.05-1 mm, or 1-5 mm, and achannel depth in the range of about 0.001-0.01 mm, 0.01-0.5 mm, 0.5-1mm, or 1-2 mm.

In accordance with an eleventh aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the sensing zone comprises an opticallytransparent area configured for measuring an optical signal that changeslevels between the absence and presence of the sample streams in thesensing zone.

In accordance with a twelfth aspect of the present disclosure, which maybe used in combination with any other aspect or combination of aspectslisted herein, the fluidic connection between the flow cell and the flowsensor is configured for a sample stream to have the same flow rateflowing through the flow cell and the flow sensor.

In accordance with a thirteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the fluidic structure comprises at least onebasic fluidic unit that comprises: a chamber configured to accommodate afluid; a venting port connected to the chamber, wherein the venting portis connected to a pneumatic pressure source, an ambient pressure, or theatmosphere pressure; a microfluidic channel connected to the chamber;and a valve on the microfluidic channel.

In accordance with a fourteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the chamber has a volume in the range of about0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml,and wherein the microfluidic channel has a cross section of a size inthe range of about 0.001-0.01 mm2, 0.01-0.1 mm2, 0.1-0.25 mm2, 0.25-0.5mm2, 0.5-1 mm2, 1-2 mm2, or 2-10 mm2.

In accordance with a fifteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the cartridge device is configured fortransferring the sample mixtures from the chamber into the flow cell toform the sample streams when an external actuation mechanism is appliedto the cartridge device, and wherein the external actuation mechanismcomprises a pneumatic pressure source.

In accordance with a sixteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, when the cartridge device is in use, the chamberis so positioned that the at least a portion of the fluid inside thechamber is pulled by gravity towards the microfluidic channel and/oraway from the venting port.

In accordance with a seventeenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, when the cartridge device is in use, the chamberhas a volume larger than the volume of the fluid accommodated thereinand an air gap exists between the venting port and the fluidaccommodated therein.

In accordance with an eighteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the valve is a passive valve that is configuredfor allowing a fluid flow to pass through the microfluidic channel whena pneumatic pressure is applied to the fluid flow and stopping the fluidflow when no pneumatic pressure is applied to the fluid flow.

In accordance with a nineteenth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the valve is a passive valve that comprises oneof the following structures: (i) a channel with a hydrophilic innersurface embedded with a patch of a hydrophobic surface, (ii) a channelwith a hydrophobic inner surface embedded with a patch of a hydrophilicsurface, (iii) an enlargement of the channel cross section along theflow direction in a channel with a hydrophilic inner surface, and (iv) acontraction of the channel cross section along the flow direction in achannel with a hydrophobic inner surface.

In accordance with a twentieth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the device further comprises a reader instrumentdevice, wherein the reader instrument device is configured forreceiving, operating, and/or actuating the cartridge device.

In accordance with a twenty-first aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the reader instrument device is configured formeasuring the optical signal at the flow cell to quantify the targetparticles in the sample.

In accordance with a twenty-second aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the reader instrument device is configured formeasuring the optical signal at the flow cell and the sensing signal atthe flow sensor to determine the concentration of the target particlesin the sample.

In accordance with a twenty-third aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, a method for analyzing target particles in asample includes applying the sample to a cartridge device, which isconfigured for collecting a predetermined sample volume into thecartridge device; transferring the cartridge device into a readerinstrument device; mixing at least a portion of the collected sample andat least a portion of a reagent to form one or more sample mixturesinside the cartridge device; forming one or more sample streams from theone or more sample mixtures in a flow cell inside the cartridge device,wherein the sample streams are formed in the flow cell without a sheathflow; measuring an optical signal from the sample streams at the flowcell to detect the target particles in the sample streams; and using thereader instrument device to analyze the measured optical signal toquantify the target particles in the sample.

In accordance with a twenty-fourth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the method includes flowing the sample streamsthrough a flow sensor that is fluidly connected to the flow cell;measuring a sensing signal from the sample streams at the flow sensor todetect the entrance of the sample streams into the flow sensor and/orthe exit of the sample streams out of the flow sensor; and using thereader instrument device to analyze the measured optical signal andsensing signal to determine the concentration of the target particles inthe sample.

In accordance with a twenty-fifth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the target particles have a size in the range of0.1-1 μm, 1-10 μm, 10-15 μm, 15-30 μm, 30-50 μm, or 50¬100 μm; andwherein the target particles have a concentration in the range of 1-100,100-1000, 1000-5000, 5000-20,000, or 20,000-50,000 target particles perμl sample steam.

In accordance with a twenty-sixth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the reagent comprises a fluorescent labelingagent that selectively labels the target particles in the sample withfluorescence, and wherein the optical signal from the sample streamscomprises fluorescence.

In accordance with a twenty-seventh aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the mixing step is performed in a fluidicstructure comprising one or a plurality of chambers, wherein eachchamber has a volume in the range of about 0.01-0.1 ml, 0.1-0.2 ml,0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml.

In accordance with a twenty-eighth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the flow cell has a width in the range of about1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm and a depth in the range ofabout 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm; and wherein thesample streams have a cross section of the same size as the flow cell.

In accordance with a twenty-ninth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the sample streams in the flow cell have a flowrate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or200-1000 μl/min when the optical signal is measured from the samplestreams.

In accordance with a thirtieth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the optical signal measured from the samplestreams at the flow cell comprises scattered light, reflected light,transmitted light, fluorescence, light absorption, light extinction, orwhite light image, or a combination thereof.

In accordance with a thirty-first aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the flow sensor comprises a fluidic channel and asensing zone on the fluidic channel, wherein the fluidic channel isfluidly connected to the flow cell to allow the sample streams to flowthrough; and wherein a sensing signal is measured when the samplestreams enter the sensing zone.

In accordance with a thirty-second aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the fluidic channel in the flow sensor has achannel width in the range of about 0.001-0.05 mm, 0.05-1 mm, or 1-5 mm,and a channel depth in the range of about 0.001-0.01 mm, 0.01-0.5 mm,0.5-1 mm, or 1-2 mm; and wherein the sample streams in the flow cell andthe flow sensor have the same flow rate.

In accordance with a thirty-third aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the sensing zone comprises an opticallytransparent area configured for measuring an optical signal that changeslevels between the absence and presence of the sample streams in thesensing zone.

In accordance with a thirty-fourth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, mixing is performed in at least one basic fluidicunit that comprises: a chamber configured to accommodate a fluid,wherein the chamber has a volume in the range of about 0.01-0.1 ml,0.1¬0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml; a venting portconnected to the chamber, wherein the venting port is connected to apneumatic pressure source, an ambient pressure, or the atmospherepressure; a microfluidic channel connected to the chamber, wherein themicrofluidic channel has a cross section of a size in the range of about0.001-0.01 mm2, 0.01-0.1 mm2, 0.1-0.25 mm2, 0.25-0.5 mm2, 0.5-1 mm2, 1-2mm2, or 2-10 mm2; and a valve on the microfluidic channel.

In accordance with a thirty-fifth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the sample mixtures are transferred from thechamber into the flow cell to form the sample streams when an externalactuation mechanism is applied to the cartridge device, and wherein theexternal actuation mechanism comprises a pneumatic pressure source.

In accordance with a thirty-sixth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, when the cartridge device is in use, the chamberis so positioned that the at least a portion of the fluid inside thechamber is pulled by gravity towards the microfluidic channel and/oraway from the venting port.

In accordance with a thirty-seventh aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, when the cartridge device is in use, the chamberhas a volume larger than the volume of the fluid accommodated thereinand an air gap exists between the venting port and the fluidaccommodated therein.

In accordance with a thirty-eighth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, at least two separate sample mixtures aretransferred into the same flow cell to form at least two separate samplestreams.

In accordance with a thirty-ninth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, a method for analyzing particles in a sampleincludes applying the sample to a cartridge device, which is configuredfor collecting a predetermined sample volume into the cartridge device;transferring the cartridge device into a reader instrument device;mixing at least a portion of the collected sample and at least a portionof a reagent to form one or more sample mixtures inside the cartridgedevice; forming one or more sample streams from the one or more samplemixtures in a flow cell inside the cartridge device, wherein at leasttwo separate sample mixtures are transferred into the same flow cell toform at least two separate sample streams without a sheath flow;measuring an optical signal from the sample streams at the flow cell todetect the target particles in the sample streams; and using the readerinstrument device to analyze the measured optical signal to quantify thetarget particles in the sample.

In accordance with a fortieth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, a portion of the collected sample is mixed with afirst reagent to form a first sample mixture and another portion of thecollected sample is mixed with a second reagent to form a second samplemixture; and wherein the two sample mixtures are separately transferredinto the flow cell to form two separate sample streams.

In accordance with a forty-first aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the two sample mixtures are separately formed ina chamber or separately transferred into a chamber before beingseparately transferred into the flow cell.

In accordance with a forty-second aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the chamber has a volume in the range of about0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml.

In accordance with a forty-third aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the sample is collected into a fluidic conduit.

In accordance with a forty-fourth aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, the fluidic conduit is closed by a valve and/orsealed by an external structure after the sample is collected into thefluidic conduit.

In accordance with a forty-fifth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, the fluidic conduit is configured for collectinga predetermine sample volume in the range of about 0.1-1 μL, 1-5 μL,5-10 μL, 10-20 μL, or 20-50 μL.

In accordance with a forty-sixth aspect of the present disclosure, whichmay be used in combination with any other aspect or combination ofaspects listed herein, at least a portion of the reagent is transferredinto the fluidic conduit to flush a portion of the collected sample intoa chamber to form a sample mixture.

In accordance with a forty-seventh aspect of the present disclosure,which may be used in combination with any other aspect or combination ofaspects listed herein, at least one sample stream comprises white bloodcells as the target particles detected in the flow cell and at leastanother sample stream comprises red blood cells and/or platelet cells asthe target particles detected in the flow cell.

The disclosure claimed is:
 1. A device for analyzing target particles ina sample, comprising: a cartridge device, wherein the cartridge devicecomprises: an inlet configured for receiving the sample into thecartridge device; a fluidic structure fluidly connected to the inlet andconfigured for mixing at least a portion of the sample with at least aportion of a reagent to form one or more sample mixtures; a flow cellfluidly connected to the fluidic structure, wherein the flow cell isconfigured for forming one or more sample streams from the one or moresample mixtures and for measuring a signal from the one or more samplestreams to detect target particles; and a flow sensor fluidly connectedto the flow cell and configured for measuring a sensing signal from thesample streams that enter the flow sensor; and a reader instrumentconfigured to measure the signal at the flow cell; wherein: the reagentcomprises a fluorescent labeling agent that selectively labels thetarget particles in the sample with fluorescence; a fluidic connectionbetween the flow cell and the flow sensor is configured for forming asample stream to have the same flow rate flowing through the flow celland the flow sensor; and the reader instrument is configured to measurethe signal at the flow cell and the sensing signal from the flow sensorto quantify the target particles in the sample.
 2. The device of claim1, wherein the flow cell is configured to form the one or more samplestreams without a sheath flow.
 3. The device of claim 1, wherein: thecartridge device comprises the reagent; and the fluidic structure isconfigured to form a sample mixture with at least a portion of thereagent after the cartridge device is received into the readerinstrument.
 4. The device of claim 1, wherein the reader instrument isconfigured to measure the signal at the flow cell and the sensing signalfrom the flow sensor to determine a concentration of the targetparticles in the sample.
 5. The device of claim 1, wherein the flowsensor comprises a sensing zone that comprises an optically transparentarea configured for measuring an optical signal that changes levelsbetween the absence and presence of the one or more sample streams inthe sensing zone.
 6. The device of claim 1, wherein at least twoseparate sample mixtures are transferred into the same flow cell to format least two separate sample streams.
 7. A device for analyzing targetparticles in a sample, comprising: a cartridge device, wherein thecartridge device comprises: an inlet configured for receiving the sampleinto the cartridge device; a fluidic structure fluidly connected to theinlet and configured for mixing at least a portion of the sample with atleast a portion of a reagent to form one or more sample mixtures; a flowcell fluidly connected to the fluidic structure, wherein the flow cellis configured for forming one or more sample streams from the one ormore sample mixtures and for measuring a signal from the one or moresample streams to detect target particles; and a flow sensor fluidlyconnected to the flow cell and configured for measuring a sensing signalfrom the sample streams that enter the flow sensor; wherein: the reagentcomprises a fluorescent labeling agent that selectively labels thetarget particles in the sample with fluorescence; the fluidic structurecomprises a chamber configured to accommodate a fluid and a venting portconnected to the chamber, the venting port being connected to apneumatic pressure source, an ambient pressure, or the atmospherepressure; and a fluidic connection between the flow cell and the flowsensor is configured for forming a sample stream to have the same flowrate flowing through the flow cell and the flow sensor.
 8. The device ofclaim 7, wherein the flow cell is configured to form the one or moresample streams without a sheath flow.
 9. The device of claim 7, wherein:the cartridge device comprises the reagent; and the fluidic structure isconfigured to form a sample mixture with at least a portion of thereagent after the cartridge device is received into a reader instrument.10. The device of claim 7, wherein the device further comprises a readerinstrument configured to measure the signal at the flow cell to quantifythe target particles in the sample.
 11. The device of claim 7, wherein:the device further comprises a reader instrument configured to measurethe signal at the flow cell, the signal being an optical signal; theflow cell comprises an optically transparent area configured formeasuring the signal from the one or more sample streams in the flowcell; and the reader instrument is configured to measure the opticalsignal at the follow cell and the sensing signal from the flow sensor toquantify the target particles in the sample.
 12. The device of claim 7,wherein the flow sensor comprises a sensing zone that comprises anoptically transparent area configured for measuring an optical signalthat changes levels between the absence and presence of the one or moresample streams in the sensing zone.
 13. A method for analyzing a sample,comprising: applying the sample to a cartridge device, comprising: aninlet configured for receiving the sample into the cartridge device; afluidic structure fluidly connected to the inlet and configured formixing at least a portion of the sample with at least a portion of areagent to form one or more sample mixtures; a flow cell fluidlyconnected to the fluidic structure, wherein the flow cell is configuredfor forming one or more sample streams from the one or more samplemixtures and for measuring a signal from the one or more sample streamsto detect target particles; and a flow sensor fluidly connected to theflow cell and configured for measuring a sensing signal from the samplestreams that enter the flow sensor; mixing at least a portion of thesample and at least a portion of the reagent to form one or more samplemixtures; forming one or more sample streams from the one or more samplemixtures in the flow cell; and measuring the signal from the one or moresample streams in the flow cell to detect the target particles; wherein:the reagent comprises a fluorescent labeling agent that selectivelylabels the target particles in the sample with fluorescence; and afluidic connection between the flow cell and the flow sensor isconfigured for forming a sample stream to have the same flow rateflowing through the flow cell and the flow sensor.
 14. The method ofclaim 13, further comprising: flowing the one or more sample streamsthrough a flow sensor that is fluidly connected to the flow cell;measuring the sensing signal from the one or more sample streams thatenter the flow sensor; and using the reader instrument device to analyzethe measured signal from the flow cell and the sensing signal from theflow sensor to quantify the target particles in the sample.
 15. Themethod of claim 13, wherein the reader instrument device analyzes themeasured signal from the flow cell to detect target cells, beads,particles, or particles bound with biological markers.
 16. The method ofclaim 13, wherein the signal at the flow cell is an optical signalselected from the group consisting of scattered light, reflected light,transmitted light, fluorescence, light absorption, light extinction, andwhite light image.
 17. The method of claim 13, wherein: the samplemixtures are transferred from the chamber into the flow cell to form thesample streams when an external actuation mechanism is applied to thecartridge device; and the external actuation mechanism comprises apneumatic pressure source.
 18. The method of claim 13, wherein, when thecartridge device is in use, the chamber is so positioned that the atleast a portion of the fluid inside the chamber is pulled by gravitytowards the microfluidic channel and/or away from the venting port. 19.The method of claim 13, wherein, when the cartridge device is in use,the chamber has a volume larger than the volume of the fluidaccommodated therein and an air gap exists between the venting port andthe fluid accommodated therein.
 20. The method of claim 13, wherein thefluidic structure further comprises a passive valve.